Glioblastoma (GBM) is the most aggressive malignant brain cancer in adults. People diagnosed with GBM have limited therapeutic options and short survival expectancies. A major problem with existing therapeutic approaches is their lack of specificity for neoplastic cells, which results in substantial treatment toxicity. Antibody-mediated specific targeting of tumor-associated antigens has been a successful strategy for cancer therapy as it limits the off-target effect of systemically infused drugs. Genetic modifications of such antibodies coupled with efficient delivery strategies can greatly improve the anti-tumor efficacy of these molecules. One such modification is bi-specific tandem single?chain antibodies (biscFv) that promote T-cell- tumor cell interactions that, in turn, kill the tumor cells. However, biscFv have short half-lives and fast clearance, necessitating frequent or continuous infusions to achieve therapeutic effect. We propose to overcome these hurdles through the generation of neural stem cells (NSCs) producing biscFv. NSCs are able to track brain tumor cells after systemic, local, and intranasal delivery, and efficiently deliver therapeutic payload to tumors sites in preclinical models of GBM. NSCs secreting biscFv can be directly mixed with autologous patients T cells for the production of a local immune response aimed at eradicating tumors. Recently, we developed and characterized a monoclonal antibody specifically targeting IL13R?2, a cell surface receptor that is selectively expressed in glioma cells, but not normal brain cells or other tissues. We demonstrated that engineered single-chain antibody retains an exclusive specificity as well as a high affinity to IL13R?2, and successfully re-targets engineered adenovirus and therapeutic CAR T cells to IL13R?2- expressing glioma cells in pre-clinical models of GBM, in vitro and in vivo. In addition to being overexpressed in the majority of GBMs, IL13R?2 expression has been associated with the highly aggressive mesenchymal subtype gene expression signature and poorer patient prognosis, all of which suggest that targeting IL13R?2- expressing glioma cells could improve GBM patient outcomes. We hypothesize that NSCs engineered to secrete bi-specific tandem IL13R?2xCD3 scFv antibody (biscFvNSCs) will promote anti-tumor immune response through the activation and engagement of T cells with GBM cells. Advancing this therapeutic for clinical application will be accomplished through R21 phase, during which we will focus on the detailed analysis and characterization of biscFvNSCs for production of functional biscFv IL13R?2xCD3 and the ability to activate T cells and elicit cytotoxic effect against IL13R?2-expressing glioma cells in vitro. During R33 phase, we will evaluate functional responses of biscFvNSCs in vivo, using immune-competent and patient-derived xenograft models of GBM. This will include the ability of biscFvNSCs to locally produce biscFv IL13R?2xCD3, engage T and glioma cells, and elicit potent anti-glioma activity. During each phase, we will achieve quantitative milestones, which will further ensure the optimization of biscFvNSCs as a new therapeutic modality for GBM treatment.